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1. |
Chloroform (CHCl3) has only one resonance in the proton spectrum. In the saturation recovery method for measuring T1, the signal is saturated by irradiation with Rf at the frequency of the signal for a period of time then the Rf is turned off and the sample allowed to recover for a period of time (t) before the spectrum is recorded. By repeating the experiment for a range of t values, the intensity of the spectrum as a function of t can be mapped out and graphed. The signal intensity recovers exponentially as function of t and T1 according to the equation St = S¥ (1-e-t/T1) Where St is the intensity of the signal at time t S¥ is the intensity of the signal at time ¥ T1 is the relaxation time of the nucleus. |
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2. |
A paramagnetic salt will broaden the spectrum of nuclei which can be relaxed by it. A suspension of cells is effectively a sample which is compartmentalised with some of the sample inside the compartments and some of the sample on the outside of the compartments. A normal NMR spectrum would simply show the sum of material inside and outside the cells. A simple experiment to differentiate signals inside and outside would be to (i) record the basic NMR spectrum and save this; (ii) add a soluble paramagnetic species which cannot penetrate the cells and again record the spectrum. This would dramatically broaden all signals which are not inside the cells and so permit them to be identified; (iii) subtract spectrum (ii) from (i) and this should subtract out the signals inside the cell from spectrum (i) leaving the signals from outside the cell clearly identified. |
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3. |
If spectra are acquired in quick succession, the signals from the species with the long relaxation time will not have sufficient time to relax between acquisitions. On the other hand, the species with the short relaxation time would be fully relaxed between acquisitions. In the first spectrum, the intensity of signals would exactly represent the concentration of species giving rise to them since the nuclei are fully relaxed prior to acquisition. For the second spectrum (4 seconds later) the nuclei with relaxation time 100 ms will be fully relaxed and will give a signal which is identical to the first spectrum. However the nuclei with long T1 will not be fully relaxed by the time the second spectrum is recorded and the signal intensity will therefore be less. The effect is re-enforced in the 3rd, 4th etc spectrum so after many acquisitions, the intensity of the signals from rapidly relaxing nuclei is relatively accurately defined however the signals from slowly relaxing nuclei will be underestimated. |
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4. |
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i) |
Fe(III) is paramagnetic so a soluble paramagnetic salt in an NMR sample will efficiently relax the nuclei in the sample. Addition of a paramagnetic salt will reduce T1 values and give rise to broad signals in the NMR spectrum. |
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ii) |
When a solution is cooled to near its freezing point, it becomes viscous and less mobile. Solutes dissolved in the solution are less mobile and tumble more slowly as the viscosity is increased. Relaxation is more efficient, relaxation times are reduced, so lines become broader as the solution becomes more viscous. |
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iii) |
Oxygen is paramagnetic and even small amounts of oxygen dissolved in the sample contributes to relaxation. So if the sample was rigorously de-oxygenated, relaxation times would increase and the lines would get sharper because the relaxation would be less efficient. |
iv) |
Relaxation is more efficient when molecular motion is slowed. Viscous solvents restrict molecular tumbling so relaxation times decrease and the lines become broader as the solution becomes more viscous. |
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5. |
The 2H spectrum is broader because the relaxation times of 2H nuclei are always shorter than for 1H. This arises because 2H has a spin of 1. Nuclei with spin other than ½ are called quadrupolar nuclei and relaxation in quadrupolar nuclei is dominated by the presence of the quadrupole. Quadrupolar relaxation, for some quadrupolar nuclei, can be so efficient that the NMR spectra are broadened to the extent that they can't be detected. |
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6. |
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i) |
The signal intensities in 13C spectra cannot usually be integrated (unless the spectra are recorded carefully under conditions that ensure that they can be properly integrated). Firstly you would expect the spectrum to contain 5 resonances - 4 for the aromatic carbons and one for the methyl carbon. The aromatic carbons should have two signals of intensity 2 (corresponding to the ortho and meta carbons and two signals of intensity 1 corresponding to the ipso and the para carbons. So ideally there should be 5 signals, 3 with intensity 1 and 2 with intensity 2. One of the factors which may be responsible for the differing signal intensity is the different relaxation times for the different carbons - the ipso carbon is not protonated and so it may well have a longer relaxation time than the other carbons. We have no indication of the time between acquisitions in this spectrum so the intensities of all signals may be underestimated (by differing amounts for each signal). If the carbons are not fully relaxed between acquisitions, then the signals in the resulting spectrum will not correctly reflect the relative numbers of nuclei giving rise to each signal. |
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ii) |
To accurately measure the integrals in the spectrum you need to ensure that all nuclei are fully relaxed between acquisitions. This can be achieved by simply waiting sufficient time between acquisitions such that the slowest relaxing nucleus is fully relaxed (usually to be safe we wait 500 approximately seconds). This can be a slow process if many acquisitions are required to obtain adequate signal. Alternatively it is common to add a small measured amount of a soluble paramagnetic salt to the sample before acquisition. The amount is critical - just sufficient to decrease relaxation times to convenient levels without broadening signals so much that the signals are difficult to observe. |